PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a successful human pathogen due to a large repertoire of virulence factors, including a variety of toxins, such as ?-hemolysin, that kill host cells and multiple secreted proteases that cleave both bacterial and host proteins. A key regulatory system for virulence factor production is the SaeRS two- component system, which activates or represses gene expression in response to an unknown signal. Recently, we identified VfrB, a conserved hypothetical protein in all gram-positive bacteria, which regulates virulence factor production consistent with altered SaeRS activity. VfrB requires a functional SaeRS to control hemolysin production and a constitutively-active SaeS bypasses the need for VfrB in ?-hemolysin expression, suggesting that VfrB plays a role in activating SaeS. In addition, we found that a mutant lacking VfrB has enhanced virulence in a mouse model of skin infection. Our further studies revealed that VfrB is a novel fatty acid kinase that works in conjunction with two uncharacterized fatty acid carrier proteins, FakB1 and FakB2, to incorporate exogenous fatty acids into the cell. Disruption of this system not only alters virulence and fatty acid uptake, but also changes growth kinetics and acetate metabolism in vitro. Interestingly, FakB2 has specificity for fatty acids not produced by this bacterium but are abundant in mammals, suggesting that VfrB and FakB may constitute a host recognition system. Based on this information, we hypothesize that VfrB in S. aureus directs carbon flow through acetate metabolism while at the same time activating the Sae system to modulate virulence. To test this hypothesis, three specific aims are proposed. Aim1 utilizes directed and global approaches to examine changes in cellular metabolism during growth, with a focus on acetate metabolism. Aim 2 uses mutagenesis to identify amino acids of VfrB, FakB1 and FakB2 that are essential for function. The mutants will be subjected to a panel of tests to examine changes in virulence factor production, protein-protein interactions and fatty acid binding. To support these studies, solving the structure of VfrB is proposed. Finally, Aim 3 will determine the stage at which VfrB controls SaeS activation, utilizing mutants and SaeRS-dependent reporters to examine the activation state of SaeRS in a mutant lacking VfrB. Aim 3 also seeks to define how VfrB regulates virulence factors in the host, and how this leads to enhanced virulence in the ?vfrB mutant. This will be addressed using a skin infection model to determine the contributions of ?-hemolysin and proteases during infection by the ?vfrB mutant. Finally, expression of the Aur protease and ?-hemolysin will be monitored in vivo using proteomics and live-animal imaging. Completion of these studies will provide insight into the activity of our recently-identified fatty acid kinase complex found in all gram-positive bacteria and will shed new light on the metabolism and regulation of virulence factors contributing to S. aureus infection.